This invention relates primarily to the development of fungal strains which express proteins at levels substantially higher than the parental strains.
For some 20 years, desired foreign proteins have been produced in microorganisms. However, having introduced the necessary coding sequence and obtained expression, much still remains to be done in order to optimise the process for commercial production One area of interest concerns strain improvement, that is to say finding or making strains of the host microorganism which enable the protein to be made in higher yields or better purity, for example.
To increase the yield, once a good expression system (eg transcription promoter) has been devised, one might envisage trying to increase the copy number of the coding sequence (although this will have the desired effect only if DNA transcription was the limiting factor), or to increase the stability of the mnRNA or to decrease the degradation of the protein. Thus, as an example of the latter approach, yeast strains (eg pep4-3) which are deficient in certain proteases have been used for producing desired foreign proteins. In another approach, the number of 2 xcexcm-based plasmids in the yeast Saccharomyces cerevisiae has been increased by introducing a FLP gene into the genome under the control of a regulated promoter, eg GAL. Upon switching to a growth medium containing galactose as the sole carbon source, plasmid copy number rises (11), but the plasmid copy number increase is uncontrolled since the GAL promoter is not repressed by REP1/REP2. This leads to reduced growth rate and thence clonal selection of ciro derivatives of the original cir+ strain (11,20).
We have mutated yeast strains by application of mutagens in order to generate mutants randomly and thereby hopefully find mutant strains which produce heterologous proteins in better yield (16,21). We have now characterised such a randomly-produced mutant which maintained a higher number of copies of the plasmid expressing the desired protein and have found that the mutation occurred in one of the genes encoding ubiquitin-conjugating enzymes, namely UBC4. The UBC4-encoded enzyme (and the closely related UBC5-encoded) enzyme are involved in degrading aberrant and short lived proteins and there was no reason to have supposed that the deletion of either of them would have enabled an increased yield of a normal, desired, protein to have been obtained.
Several genes encoding ubiquitin conjugating enzymes (UBC) have been implicated in the bulk protein degradation and in the stress response of yeast. UBC1, UBC4 and UBC5 act together to mediate important functions for cell growth and cell viability (2,3). Yeast strains with a mutation in a single gene are viable and have similar growth rates to the parental strains, but ubc4/ubc5 double mutants have reduced growth rates and are sensitive to amino acid analogues, while a triple mutant is inviable, indicating that their activities overlap. The UBC4 and UBC5 genes are closely related and the two coding DNA sequences share 77% identical residues, while the predicted amino acid sequences of the two proteins show 92% identical residues (3). Because of the near identity of the Ubc4 and Ubc5 proteins (hereafter abbreviated to Ubc4p and Ubc5p) it has been suggested that UBC4 could complement for the loss of function of the ubc5 mutant and vice versa (3). This would explain why the dramatic reduction in growth rate was only observed in ubc4/ubc5 double mutants. Pulse chase experiments have indicated that Ubc4p and Ubc5p are responsible for the degradation of short-lived and abnormal proteins, but that the turnover of these proteins was only reduced in strains with the ubc4/ubc5 double mutation. It was not reduced in strains with single ubc4 or ubc5 mutations (3). This reference, therefore, suggested that the use of single ubc4 and ubc5 mutant fungal strains would not be beneficial.
Structurally, all known UBC genes encode a conserved domain (known as the UBC domain) of approximately 16kDa containing the conserved conjugating cysteine (1,22). Transfer of activated ubiquitin results in the covalent attachment of the C-terminus of ubiquitin via a thioester bond to the cysteine residue. UBC genes have been divided into different classes (reviewed in 22). Class I UBC genes are composed almost exclusively of the conserved UBC domain, class II and class III UBC genes have C-terminal or N-terminal extension, respectively, while class IV UBC genes have both C- and N-terminal extensions (22).
The fungal genome is composed of chromosomes, extrachromosomal copies of chromosomal genes, eg nucleosomes, and occasionally stable extrachromosomal elements. These extrachromosomal elements have developed a benignly parasitic relationship with their host, where they successfully balance the theft of cellular resource for the replication and segregation of the element, while not compromising the fitness of the host. General reviews of fligal extrachromosomal elements are covered by references 5 and 6, while the DNA plasmids of the yeasts Saccharomyces species are covered by references 7 and 8 and Kluyveromyces species are covered by reference 9.
The 2 xcexcm plasmids of Saccharomyces species are extrachromosomal DNA species which have evolved mechanisms to ensure their long term autonomous survival without any associated phenotype. The 2 xcexcm plasmid resides in the nucleus and is packaged into chromatin. The plasmid origin of replication acts as an autonomously replicating sequence, while other sequences ensure the maintenance of a controlled high copy number and allow the plasmid to partition uniformly into the daughter cells at mitosis. The plasmid is not required for normal mitotic growth and does not provide the host with any selective advantage since Saccharomyces species devoid of 2 xcexcm plasmid, denoted as ciro, grow only slightly faster than their 2 xcexcm plasmid containing, or cir+, parents.
The 2 xcexcm plasmid is a double stranded circular plasmid of approximately 6,318 bp, comprising two unique regions of 2,774 and 2,346 bp separated by a pair of exact inverted repeats, each 599 bp long (10). In vivo the monomeric plasmid exists as an equal mixture of the two inversion isomers (A and B) that form following site specific recombination between the two inverted repeats. The 2 xcexcm plasmid has four open reading frames known as FLP (also known as A), REP1 (also known as B), REP2 (also known as C) and RAF (also known as D). The plasmid also contains a region, located between RAF and the origin of replication, called STB or REP3, which is composed of a series of imperfect 62 bp repeat elements This element is required in cis, along with the trans acting elements, REP1 and REP2, to enable efficient partitioning of the plasmid between the mother and the daughter cell.
The 2 xcexcm plasmid copy number is also indirectly under the control of chromosomal genes, since it is known that 2 xcexcm plasmid copy number does vary between different Saccharomyces cerevisiae strains and because the chromosomal recessive mutation, known as nib1, results in clonal lethality due to uncontrolled amplification of 2 xcexcm plasmid copy number (39). Yeast strains carrying the nib1 mutation resemble engineered yeast strains where FLP gene expression is galactose induced. The involvement of proteins of the fungal ATP-dependent ubiquitin protein degradation pathway in the regulation of fungal plasmid copy number is not described in the art. Nor is it disclosed that genes of the fungal ATP-dependent ubiquitin protein degradation pathway can be manipulated to control fungal plasmid copy number.
Although the 2 xcexcm plasmid is a very common genetic component of Saccharomyces cerevisiae, other yeast strains are known to contain identifiable DNA plasmids, notably the pSR1 and pSB3 plasmids (6,251 bp and 6,615 bp) of Zygosaccharomyces rouxii, the pSB1 and pSB2 plasmids (6,550 bp and 5,415 bp) of Zygosaccharomyces baijii, the pSM1 plasmid (5,416) of Zygosaccharomyces fermentati and the pKD1 plasmid (4,757 bp) of Kluyveromyces drosophilarum (9). The most striking feature of all these plasmids is their resemblance to the Saccharomyces cerevisiae 2 xcexcm plasmid. Each plasmid is circular, double stranded DNA and is composed of two approximately equally sized halves separated by inverted repeat sequences. Each plasmid contains a single Autonomously Replicating Sequence (ARS) close to one of the inverted repeat sequences and three or four open reading frames, one of which encodes a recombinase which catalyses recombination between the inverted repeats.
A Saccharomyces cerevisiae plasmid is considered to be xe2x80x9c2 xcexcm-basedxe2x80x9d if it contains at least one of the 2 xcexcm plasmid elements (ARS, inverted repeat sequences or 2 xcexcm open reading frames), especially the ARS.
One aspect of the present invention provides a process of producing a fungal cell derived product, comprising (i) providing a fungal cell having a plasmid, the plasmid comprising a functional coding sequence for a protein, and the fungal cell having a modified level of Ubc4p or Ubc5p (hereinafter, generically known as Ubcp activity), and (ii) culturing the cell to produce the fungal cell derived product.
Preferably the fungal cell derived product is a desired protein encoded by the said coding sequence, and the said modified level of Ubcp activity is lower than normal for the cell. This can be tested in vivo by assaying for the rate of abnormal protein turnover (3). The level of Ubcp (Ubc4p and/or Ubc5p) activity may be reduced to at most 50%, 40%, 30%, 20%, 10% or 1% of the wild-type level. Preferably, the cell has a minimal Ubc4p or Ubc5p activity. The cell should not, however, have a low level of both Ubc4p and Ubc5p, since its growth rate will generally be too low to be useful.
The reduction in Ubcp activity can be achieved in any one of a variety of ways. Firstly the cell can produce a compound which interferes with the binding of the UBC-encoded product to its receptor. Hence, a construct may be provided in the cell to express a polypeptide which competes for the binding of Ubc4p or Ubc5p to its target. This will facilitate a reduction in the effective Ubc4p or Ubc5p activity. This may be done by over-expressing the UBC domain encoded by UBC4 or UBC5 described above. It will be important to ensure that the over-expressed UBC domain encoded by UBC4 or UBC5 does not have any intrinsic Ubc4p or Ubc5p activity of its own, since this might actually contribute to the overall Ubc4p or Ubc5p activity. This may be achieved, by site directed mutagenesis, by removing or replacing (for example with an alanine) the cysteine which acts as the acceptor site for the ubiquitin within the UBC domain of UBC4 (or 5) or other conserved amino acids within the UBC domain. Over expression of the inactive UBC domain of UBC4 (or 5) may be achieved from its own endogenous promoter, or from any other convenient promoter. The construct may be integrated into the chromosome or episomal.
Alternatively, in order to achieve a reduced level of Ubcp activity, the endogenous UBC gene may be modified such that substantially no protein is produced therefrom or such that any protein produced therefrom has a reduced level of Ubc4p or Ubc5p activity. Thus, for example, the UBC gene may be deleted (either in a regulatory region or in the coding region or both) such that no polypeptide is produced or a mutant (defective) polypeptide product is produced. (By xe2x80x9cregulatory regionxe2x80x9d, we include parts of the genome acting on the UBC gene indirectly, for example a gene producing a UBC gene activator.) Deletion of all or part of the UBC open reading frame (14) is preferred, as this will reduce or abolish Ubcp activity and generate a non-reverting mutant fungal strain. Alternatively, the activity can be reduced or abolished by classical mutagenesis procedures, whereby the DNA sequence of the UBC gene is mutated in such a way as to produce point mutations or deletions which modify and/or disrupt the normal amino acid sequence of the Ubcp. If a mutant Ubcp polypeptide is produced, it may be unstable (ie be subject to increased protein turnover relative to the native protein); or unable to conjugate ubiquitin, or unable to deliver bound ubiquitin to its substrate.
For example, the UBC gene may be modified such that the ubiquitin-accepting cysteine in any protein produced therefrom is absent or of reduced ubiquitin-accepting activity, for example due to alterations in the amino acid residues surrounding or otherwise interacting with the cysteine, as noted above in the context of producing competitive (but inactive) polypeptide. Alternatively, the UBC gene may be modified such that the capacity of any mutant protein produced therefrom is unable to interact with or has reduced affinity for the E1 ubiquitin donor (product of the UBA1 or UBA2 genes). Alternatively, the UBC gene may be modified such that the capacity of any mutant protein produced therefrom to interact with the final ubiquitin acceptor and/or the Ubiquitin ligase (E3) enzyme is absent or reduced. Specifically, mutations (deletion, insertions or substitutions) within the first 21 amino acids of the primary sequence and the first xcex1 helix (residues 3-13) of Ubc4p and Ubc5p (29) are preferred as the latter have been implicated in binding of Ubc2p, a related protein, to the ubiquitin protein ligase Ubr1p (33). Especially preferred are mutations affecting the glutamic acid at position 10 (Glu-10) within the primary sequence of Ubc4p and Ubc5p, particularly replacement by lysine (Glu10Lys) or arginine (Glu10Arg).
Alternatively a different promoter may be used to control expression of the UBC gene; such a promoter may be regulatable. For example, it may be inducible, as are promoters of the galactose utilisation pathway, or derepressed by the removal of an inhibitor, as are promoters of the acid phosphatase group.
Site directed mutagenesis or other known techniques can be employed to create single or multiple mutations, such as replacements, insertions, deletions, and transpositions, as described in reference 23. Suitable mutations include chain termination mutations (clearly stop codons introduced near the 3xe2x80x2 end might have insufficient effect on the gene product to be of benefit; the person skilled in the art will readily be able to create a mutation in, say, the 5xe2x80x2 three quarters of the coding sequence), point mutations that alter the reading frame, small to large deletions of coding sequence, mutations in the promoter or terminator that affect gene expression and mutations that de-stabilize the mRNA. Specific mutations can be introduced by an extension of the gene disruption technique known as gene transplacement (24).
Generally one uses a selectable marker to disrupt a gene sequence, but this need not be the case, particularly if one can detect the disruption event phenotypically. In many instances the insertion of the intervening sequence will be such that a stop codon is present in frame with the UBC sequence and the inserted coding sequence is not translated. Alternatively the inserted sequence may be in a different reading frame to UBC.
A third principal way to achieve a reduction of Ubcp activity is for the cell to produce UBC antisense mRNA. This may be achieved in conventional ways, by including in the cell an expression construct for an appropriate sequence. UBC antisense mRNA may be produced from a constitutive or regulated promoter system (eg promoters of the galactose catabolic pathway), thereby facilitating a reduction in translatable UBC mRNA. Use of the regulated UBC antisense mRNA also allows for control of the ubiquitin-dependent protein degradation pathway by the addition or removal of the activator.
Fungal cells useful in the methods of the invention include the genera Pichia, Saccharomyces, Zygosaccharomyces, Kluyveromyces, Candida, Torulopsis, Hansenula (now reclassified as Pichia), Schizosaccharomyces, Citeromyces, Pachysolen, Debaromyces, Aspergillus, Metschunikowia, Rhodoporidum, Leucosporidum, Botryoascus, Endomycopis, Trichoderma, Cephalosporium, Humicola, Mucor, Neurospora and the like. Preferred genera are Pichia, Saccharomyces, Zygosaccharomyces and Kluyveromyces. Examples of Saccharomyces sp. are Saccharomyces cerevisiae, Saccharomyces italicus, Saccharomyces diastaticus and Zygosaccharomyces rouxii. Examples of Kluyveromyces sp. are Kluyveromyces fragilis and Kluyveromyces lactis. Examples of Hansenula sp. are Hansenula polymorpha (now Pichia angusta), Hansenula anomala (now Pichia anomala) and Pichia capsulata. An example of a Pichia sp. is Pichia pastoris. Examples of Aspergillus sp. are Aspergillus niger and Aspergillus nidulans. Yarrowia lipolytica is an example of a suitable Yarrowiva species.
Preferred are yeast strains, and of these Saccharomyces cerevisiae is the particularly preferred host. The yeast strains used can be any haploid or diploid strain of Saccharomyces cerevisiae, but in the case of diploid strains it is preferred that the activity of the Ubcp enzyme from both copies of the UBC gene is reduced or abolished.
A number of species have been shown to have homologues of Saccharomyces cerevisiae UBC4 and UBC5 genes. UBC4 and UBC5 homologues have been described in Homo sapiens (34) , Drosophila melanogaster (35), Caenorhabditis elegans (36), Arabidopsis thaliana (37), Schizosaccharomyces pombe and Candida albicans (38). The Drosophila melanogaster and Caenorhabditis elegans homologues, UbcD1 and ubc-2, respectively, have also been shown to have Ubcp activity. It can be seen that a homologue need not be termed UBC4 or UBC5; equally, a gene which is called UBC4 or UBC5 need not be a homologue.
Of the known UBC4/UBC5 homologues in the literature, the similarity of the various proteins can be calculated by aligning the primary amino acid sequences. A suitable program is the Megalign Program, Lasergene, DNASTAR Inc, 1228 South Park Street, Madison, Wis. 53715, USA. Using such a program the calculated percentage similarity ranges from 75.7% to 97.3. These values are very high and reflect the highly conserved nature of the Ubc proteins. The highly conserved cysteine residue in the active site occurs at position 193 in the consensus sequence.
Of the other Ubc proteins, the calculated percentage similarity between them and to Saccharomyces cerevisiae Ubc4p and Ubc5p ranged from 24.2% to 63.5%. Proteins homologous to the Saccharomyces cerevisiae Ubc4p and Ubc5p can therefore be defined as any Class I (as defined by Jentsch, 1992, reference 22) ubiquitin conjugating enzyme, which possesses 66.7% or greater primary amino acid sequence similarity to Saccharomyces cerevisiae Ubc4p or Ubc5p, as defined by the Megalign program. A gene is deemed to be homologous to S. cerevisiae UBC4 or UBC5 if it encodes such an enzyme.
A number of species have also been shown to possess Ubcp activity. As stated previously ubc4/ubc5 double mutants of Saccharomyces cerevisiae have increased doubling time, reduced resistance to amino acid analogues and reduced resistance to heat shock. It is known that the Drosophila UBC1 protein, encoded by the UbcD1 gene, which is 79.6% and 80.3% similar to Saccharomyces cerevisiae Ubc4p and Ubc5p respectively, can reverse the phenotypes of a yeast with no Ubc4p or Ubc5p activity when placed downstream of the UBC4 promoter (35). Similarly it is also known that the Caenorhabditis protein ubc-2, encoded by the ubc-2 gene, which is 78.2% and 78.9% similar to Saccharomyces cerevisiae Ubc4p and Ubc5p respectively, has the same properties (36). This is therefore a functional test of whether a protein from an unknown source has Ubc4p or Ubc5p activity. It can also be seen that, for the examples of doubling time and survival rate after 24 hrs at 38xc2x0 C., the single ubc4 or ubc5 mutant strains described by Seufert and Jentsch (3,36) have similar characteristics to the wild-type strain. The Ubcp activity of an unknown Ubc protein, or a mutant form of a known Ubc protein, relative to the natural Saccharomyces cerevisiae Ubc4p or Ubc5p, can be determined by its relative ability to return the doubling time or survival rate after 24 hrs at 38xc2x0 C. (as described in references 3 or 36), of a double ubc4/ubc5 mutant strain to normal for a wild type or single ubc4 or ubc5 mutant Saccharomyces cerevisiae strain once the unknown or mutant Ubc protein has been integrated into the Saccharomyces cerevisiae genome under the control of the endogenous UBC4 or UBC5 promoter, preferably as a single copy integration at the endogenous UBC4 or UBC5 locus by procedures already described in the literature (36).
In a preferred aspect of the invention, the level of Ubc4p or Ubc5p activity is reduced. This has been found to increase the copy number of an expression plasmid in the cell, and to cause an increased level of expression of a desired protein expressed from the plasmid. Conversely, increasing the level of Ubc4p or Ubc5p activity will reduce the level of expression of the protein, which may be desirable in some circumstances, for, example where the plasmid-encoded protein inhibits production of the desired protein.
The term xe2x80x9cdesired proteinxe2x80x9d is used herein in the normal sense to mean any protein (or other polypeptide) which is desired in a given process at a higher level than the one at which the fungal cell would, without human intervention, produce it. The desired protein may be endogenous to the species in question, for example it may be an enzyme which is normally produced by the host cell. Usually, however, the protein is heterologous to the host cell. The protein may perform its required task in the host cell or host cell culture without being extracted. Usually, however, the protein is extracted from the cell culture and purified to some extent for use elsewhere. The protein may be a viral, microbial, fungal, plant or animal protein, for example a mammalian protein. Preferably, it is a human protein, for example albumin, immnunoglobulin or a fragment thereof (such as an Fab fragment or single chain antibody), (haemo-)globin, blood clotting factors (such as factors II, VII, VIII, IX), interferons, interleukins, xcex1I-antitrypsin, insulin, calcitonin, cell surface receptors, fibronectin, pro-urokinase, (pre-pro)chymosin, antigens for vaccines, t-PA, tumour necrosis factor, erythropoietin, G-CSF, GM-CSF growth hormone, plateletderived endothelial cell growth factor, and enzymes generally, such as glucose oxidase and superoxide dismutase. The protein is, of course, not Ubc4p or Ubc5p itself, nor a fusion of either Ubc4p or Ubc5p in which the Ubc4p or Ubc5p performs its natural function.
The desired protein, if it is to be purified from the fungal cell culture, may be obtained by any technique suited to that protein. For example, albumin may be purified from a Saccharoznyces, Kluveromyces or Pichia cell culture according to the techniques disclosed in WO96/37515, EP-625 202 or EP-464 590, respectively.
Our work has principally involved human albumin, although there is no reason to suppose that the process of the invention is applicable only to this protein, especially since the invention has also been shown to be advantageous in the expression of human haemoglobin.
The term xe2x80x9chuman albuminxe2x80x9d is used herein to denote material which is indistinguishable from human serum albumin or which is a variant or fragment thereof. By xe2x80x9cvariantxe2x80x9d we include insertions, deletions and substitutions, either conservative or non-conservative, where such changes do not substantially alter the oncotic, useful ligand-binding or immunogenic properties or albumin. For example we include naturally-occurring polymorphic variants of human albumin or human albumin analogues disclosed in EP-A-322 094. Generally, variants or fragments of human albumin will have at least 50% (preferably at least 80%, 90% or 95%) of human serum albumin""s ligand binding activity (for example bilirubin-binding) and at least 50% (preferably at least 80%, 90% or 95%) of human serum albumin""s oncotic activity, weight for weight.
The desired protein coding region is preferably contained within a hybrid plasmid comprising a promoter sequence, a DNA coding sequence which is under the transcriptional control of the promoter, a leader sequence directing the secretion of the protein and a DNA sequence containing a eukaryotic transcription termination signal, which plasmid is then maintained as an extrachromosomal DNA sequence or is integrated into one or more chromosomes of the host organism.
Suitable promoters for the expression of the protein include those associated with the phosphoglycerate kinase (PGK1) gene, galactokinase (GAL1) and uridine diphosphoglucose 4-epimerase (GAL10) genes, iso-1-cytochrome c (CYC1), acid phosphatase (PHO5), alcohol dehydrogenase genes (ADH1and ADH2) and MFxcex1-1. The preferred promoters are the glycerol-3-phosphate dehydrogenase (GPD1), described in EP 424 117, and the protease B (PRB1) promoter, described in EP-431 880 B1.
Suitable transcription termination sequences can be the 3xe2x80x2 flanking equence of the eukaryotic gene which contains proper signals for transcription termination and polyadenylation in the fungal host, or those of the gene naturally linked to the expression control sequence, or those associated with the phosphoglycerate kinase (PGK1) or the iso-1-cytochrome c (CYC1) gene. The preferred transcription termination sequence is from the alcohol dehydrogenase gene (ADH1).
Suitable secretory leader sequences are, for example, the natural human serum albumin leader sequence, the leader sequence from the Saccharomyces cerevisiae MFxcex1-1 leader sequence, the Kluyveromyces lactis killer toxin leader, a fusion between the natural human serum albumin leader and the Saccharomyces cerevisiae MFxcex1-1 leader sequence, or a fusion between the Kluyveromyces lactis killer toxin leader and the Saccharomyces cerevisiae MFxcex1-1 leader sequence, or conservatively modified variations of these sequences, as described in WO 90/01063.
Hybrid plasmids may also be used which, apart from the expression control sequence, the heterologous gene sequence and the transcription termination sequence, contain additional sequences which are non-essential or less important for the function of the promoter, ie for the expression of the desired polypeptide, but which perform important functions in, for example, the propagation of the cells transformed with the said hybrid plasmids. The additional DNA sequences may be derived from prokaryotic and/or eukaryotic cells and may include chromosomal and/or extra-chromosomal DNA sequences. For example, the additional DNA sequences may stem from (or consist of) plasmid DNA, such as bacterial, yeast or higher eukaryotic chromosomal DNA. Preferred hybrid plasmids contain additional DNA sequences derived from bacterial plasmids, especially Escherichia coli plasmid pBR322 or related plasmids, bacteriophage, yeast 2 xcexcm plasmid, and/or yeast chromosomal DNA.
In the preferred hybrid plasmids for the expression of the heterologous polypeptide, the additional DNA sequences carry a yeast replication origin and a selective genetic marker for yeast. Hybrid plasmids containing a yeast replication origin, eg an autonomously replicating segment (ARS), are extrachromosomally maintained with the yeast cells after transformation and are autonomously replicated upon mitosis. Hybrid plasmids containing sequences homologous to the yeast 2 xcexcm plasmid DNA can be as well. These hybrid plasmids may be integrated by recombination into yeast 2 xcexcm plasmids already present within the cell or may replicate autonomously. The integration vectors of EP-A-251 744 or the xe2x80x9cdisintegrationxe2x80x9d vectors of EP-A-286 424 may be used.
Advantageously, the additional DNA sequences which are present in the hybrid plasmids also include a replication origin and a selective marker for the bacterial host, especially Escherichia coli, and a selectable marker for the final fungal host. There are useful features which are associated with the presence of an Escherichia coli replication origin and an Esclerichia coli marker in a yeast hybrid plasmid. Firstly, large amounts of hybrid plasmid DNA can be obtained by growth and amplification in Escherichia coli and, secondly, the construction of hybrid plasmids is conveniently done in Escherichia coli making use of the whole repertoire of cloning technology based on Escherichia coli. Escherichia coli plasmids, such as pBR322 and the like, contain both Escherichia coli replication original and Escherichia coli genetic markers conferring resistance to antibiotics, for example tetracycline and ampicillin, and are advantageously employed as part of the yeast hybrid vectors. The selective fungal marker may be any gene which facilitates the selection of transformants due to the phenotypic expression of the marker. Suitable markers are particularly those expressing antibiotic resistance or, as in the case of auxotrophic yeast mutants, genes which complement host lesions. Corresponding genes confer, for example, resistance to the antibiotic cycloheximide or provide for prototrophy in an auxotrophic yeast mutant, for example the URA1, URA3, LEU2, HIS3, HIS4, TRP5, TRP1 and LYS2 genes.
It has been demonstrated that ftingal cells of the genera Pichia, Saccharomyces, Kluyveromyces, Yarrowia and Hansenula can be transformed by enzymatic digestion of the cell walls to give spheroplasts; the spheroplasts are then mixed with the transforming DNA and incubated in the presence of calcium ions and polyethylene glycol, then transformed spheroplasts are regenerated in regeneration medium. The regeneration medium is prepared in such a way as to allow regeneration and selection of the transformed cells at the same time.
Since the yeast genes coding for enzymes of nucleic acid or amino acid biosynthetic pathways are generally used as selection markers, the regeneration is preferably performed in yeast minimal medium. Methods for the transformation of Saccharomyces cerevisiae are taught generally in EP 251 744, EP 258 067, WO 90/01063 and by Hinnen et al (4), all of which are incorporated herein by reference.
Hence, in its broadest aspect, the invention provides the use of a means to vary UBC4 or UBC5 function in a fungal cell to control the copy number of a plasmid in that cell.